BPI and its congeners as radiation mitigators and radiation protectors

ABSTRACT

Described herein is a method of mitigating, in a subject (individual), tissue injury resulting from exposure to radiation (accidental/unintentional or intentional, such as therapeutic), chemoradiotherapy, disease, toxin, or drug or biologic mediated therapy.

RELATED APPLICATIONS

This application is a continuation of Ser. No. 14/009,201, filed Dec.17, 2013, which is a national stage filing under 35 U.S.C. §371 ofinternational application PCT/US2012/032288, filed Apr. 5, 2012, whichwas published under PCT Article 21(2) in English, and claims the benefitunder 35 U.S.C. §119(e) of U.S. provisional application Ser. No.61/471,896, filed Apr. 5, 2011, the disclosures of which areincorporated by reference herein in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under grant number U19AI067751 awarded by The National Institutes of Health and grant numbersHR0011-08-1-0011 and HR0011-12-1-0014 awarded by The Defense AdvancedResearch Projects Agency. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Exposure of humans to radiation can cause serious harm, even death.Exposure can be accidental, resulting, for example, from a radiationleak at a nuclear power plant. Exposure also can be intentional,resulting, for example, from an act of terror. The most commoncircumstance of radiation exposure results from medical interventions,such as for the treatment of cancer. Radiation in this context can belocal or systemic. When applied locally, radiation can nonetheless causeunwanted injury to healthy tissue in the pathway of the radiation. Whenapplied systemically (i.e., total body irradiation), low doses can leadto bone marrow damage and gastrointestinal tract toxicity. High doses oftotal body irradiation can lead to permanent hone marrow damage, gut andlung toxicity, and sometimes death. There is a need for effectivetreatments which protect healthy tissues and mitigate the acute andchronic effects of exposure to ionizing radiation.

SUMMARY OF THE INVENTION

Described herein is a method of mitigating, in a subject (individual),tissue injury resulting from exposure to radiation(accidental/unintentional or intentional, such as therapeutic),chemoradiotherapy or disease. The method comprises administering to thesubject (individual), referred to as a subject in need thereof,bactericidal/permeability increasing protein (BPI), a (at least one, oneor more) BPI congener or both BPI and a BPI congener in an amountsufficient to reduce (partially or completely) the effects of exposure,thereby mitigating tissue injury resulting from radiation exposure inthe subject. In certain embodiments, the radiation exposure results fromaccidental exposure to radiation, such as occurs in the event of anuclear plant failure, or intentional exposure to radiation, such astherapeutic radiation, chemoradiotherapy or radiotherapy. Tissue injurycan be, for example, injury to hematopoietic tissue (e.g., bone marrow)or injury to the gastrointestinal (GI) tract. In a particularembodiment, the tissue injury is hematopoietic toxicity. BPI congenersused in the method include, but are not limited to, rBPI₂₁, rBPI₂₃,rBPI₅₀, rBPI(10-193)ala¹³² and a N-terminal fragment of BPI having anapproximate molecular weight of from about 20 kD to about 25 kD.

In certain embodiments, BPI and/or its congeners is administered between1 day before exposure of the subject to radiation and 2 days (48 hours)after exposure of the subject to the radiation. BPI and/or its congenerscan be administered orally, intravenously, or subcutaneously.

In some embodiments, the method of mitigating tissue injury in a subjectresulting from exposure to radiation (accidental/unintentional orintentional, such as therapeutic), chemoradiotherapy or disease furthercomprises administering an (at least one, one or more) antibiotic to thesubject (a subject in need thereof). The antibiotic can be, for example,a quinolone antibiotic, such as an antibiotic selected from the groupconsisting of moxifloxacin, ciprofloxacin, levofloxacin, garenoxacin anddelafloxacin.

In another aspect, the method is a method of mitigating hematopoietictoxicity in a subject (individual) resulting from exposure to radiation(accidental/unintentional or intentional, such as therapeutic),chemoradiotherapy, disease, toxin or drug or biologic mediated therapy.The method comprises administering to the subject (individual), referredto as a subject in need thereof, bactericidal/permeability increasingprotein (BPI), a (at least one, one or more) BPI congener or both BPIand a BPI congener in an amount sufficient to mitigate (partially orcompletely) the hematopoietic toxicity of the subject. BPI congenersused in the method include, but are not limited to, rBPI₂₁, rBPI₂₃,rBPI₅₀, rBPI(10-193)ala¹³² and a N-terminal fragment of BPI having anapproximate molecular weight of from about 20 kD to about 25 kD.

In certain embodiments, BPI and/or its congeners is administered between1 day before exposure of the subject to radiation and 2 days (48 hours)after exposure of the subject to the radiation. BPI and/or its congenerscan be administered orally, intravenously, or subcutaneously.

In some embodiments, the method of mitigating hematopoietic toxicity ina subject (individual) resulting from exposure to radiation(accidental/unintentional or intentional, such as therapeutic),chemoradiotherapy, disease, toxin, or drug or biologic mediated therapyfurther comprises administering an (at least one, one or more)antibiotic to the subject (a subject in need thereof). The antibioticcan be, for example, a quinolone antibiotic, such as an antibioticselected from the group consisting of moxifloxacin, ciprofloxacin,levofloxacin, garenoxacin and delafloxacin.

A further embodiment is a method for bone marrow recovery in a subject(individual), the method comprising: administering to the subject(individual), referred to as a subject in need thereof,bactericidal/permeability increasing protein (BPI), a (at least one, oneor more) BPI congener or both BPI and a BPI congener in an amountsufficient for bone marrow recovery in the subject. BPI congeners usedin the method include, but are not limited to, rBPI₂₁, rBPI₂₃, rBPI₅₀,rBPI(10-193)ala¹³² and a N-terminal fragment of BPI having anapproximate molecular weight of from about 20 kD to about 25 kD.

In certain embodiments, BPI and/or its congeners is administered between1 day before exposure of the subject to radiation and 2 days (48 hours)after exposure of the subject to the radiation. BPI and/or its congenerscan be administered orally, intravenously, or subcutaneously.

In certain embodiments, the subject has a deficiency in one or morehematopoietic cell types or lineages. For example, a subject may have ahematopoietic deficiency such as lymphopenia, myelopenia, leukopenia,neutropenia, erythropenia, megakaryopenia, a deficiency in platelets, adeficiency in monocytes, a deficiency in lymphocyctes, a deficiency inerythrocytes, deficiency in neutrophils, a deficiency in T cells, adeficiency in granulocytes, and/or a deficiency in dendritic cells. Thedeficiency in one or more hematopoietic cell types or lineages mayresult from exposure to radiation, chemoradiotherapy, radiotherapy,disease, toxin, or drug or biologic mediated therapy.

In some embodiments, the method of bone marrow recovery in a subject(individual) resulting from exposure to radiation(accidental/unintentional or intentional, such as therapeutic),chemoradiotherapy, disease, toxin or drug or biologic mediated therapyfurther comprises administering an (at least one, one or more)antibiotic to the subject (a subject in need thereof). The antibioticcan be, for example, a quinolone antibiotic, such as an antibioticselected from the group consisting of moxifloxacin, ciprofloxacin,levofloxacin, garenoxacin and delafloxacin.

A further embodiment is a method for stimulating hematopoiesis in asubject (individual), the method comprising: administering to thesubject (individual), referred to as a subject in need thereof,bactericidal/permeability increasing protein (BPI), a (at least one, oneor more) BPI congener or both BPI and a BPI congener in an amountsufficient to stimulate hematopoiesis in the subject. BPI congeners usedin the method include, but are not limited to, rBPI₂₁, rBPI₂₃, rBPI₅₀,rBPI(10-193)ala¹³² and a N-terminal fragment of BPI having anapproximate molecular weight of from about 20 kD to about 25 kD.

In certain embodiments, the subject has a deficiency in one or morehematopoietic cell types or lineages. The hematopoietic deficiency canbe, for example, lymphopenia, myelopenia, leukopenia, neutropenia,erythropenia, megakaryopenia, a deficiency in platelets, a deficiency inmonocytes, a deficiency in lymphocyctes, a deficiency in erythrocytes,deficiency in neutrophils, a deficiency in T cells, a deficiency ingranulocytes, and/or a deficiency in dendritic cells. The deficiency inone or more hematopoietic cell types or lineages results, for example,from exposure to radiation, chemoradiotherapy, radiotherapy, disease,toxin or drug or biologic mediated therapy. BPI congeners used in themethod include, but are not limited to, rBPI₂₁, rBPI₂₃, rBPI₅₀,rBPI(10-193)ala¹³² and a N-terminal fragment of BPI having anapproximate molecular weight of from about 20 kD to about 25 kD.

In certain embodiments, BPI and/or its congeners is administered between1 day before exposure of the subject to radiation and 2 days (48 hours)after exposure of the subject to the radiation. BPI and/or its congenerscan be administered orally, intravenously, or subcutaneously.

In certain embodiments, the method for stimulating hematopoiesis in asubject (individual) further comprises administering an (at least one,one or more) antibiotic to the subject (a subject in need thereof). Theantibiotic can be, for example, a quinolone antibiotic, such as anantibiotic selected from the group consisting of moxifloxacin,ciprofloxacin, levofloxacin, garenoxacin and delafloxacin.

As described herein, in certain embodiments, the method comprisesadministering (a) bactericidal/permeability increasing protein (BPI), a(at least one, one or more) BPI congener or both BPI and a BPI congenerand (b) an (at least one, one or more) antibiotic to a subject(individual) in need thereof. In those embodiments, thebactericidal/permeability increasing protein (BPI), BPI congener or bothBPI and a BPI congener and the antibiotic can be administeredsimultaneously (together) or sequentially. In those instances in whichthe bactericidal/permeability increasing protein (BPI), BPI congener orboth BPI and a BPI congener and the antibiotic are administeredsimultaneously, they can be administered in one composition or inseparate compositions. In those instances in which thebactericidal/permeability increasing protein (BPI), BPI congener or bothBPI and a BPI congener and the antibiotic are administered sequentially,they can be administered in either order and need to be administeredsufficiently close in time that they have the desired mitigating effect.

The subject (individual) is an animal, typically a mammal. In oneaspect, the subject is a dog, a cat, a horse, a sheep, a goat, a cow ora rodent. In important embodiments, the subject is a human. In any ofthe foregoing embodiments, the subject is not otherwise in need oftreatment with BPI and/or its congeners. In some such embodiments, thesubject does not have an infectious disease.

These and other aspects of the inventions, as well as various advantagesand utilities will be apparent with reference to the DetailedDescription. Each aspect of the invention can encompass variousembodiments as will be understood.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Changes in circulating levels of neutrophils (ANC) and platelets(PLT), monocyte surface levels of mCD14 and TLR4, plasma levels ofendotoxin, BPI, and IL-6, and incidence of fever are observed afterhuman myeloablative HSCT. (A) Severe neutropenia (n=46) andthrombocytopenia occurred (n=48, nadir D7). Data represent geometricmeans+SEM of log transformed values labeled in original units. (B)Plasma endotoxin was evaluated in 18 patients by endotoxin activityassay (EAA) and reported in EAA units at baseline (B; n=17) and for D0(n=17), D7 (n=10), D14 (n=15), D21 (n=15) and D28 (n=3) aftermyeloablation. The horizontal dashed line (at 0.4 EA units) indicateslower limit of detection (LLD). Plasma BPI concentrations in pg/mL wereassessed by ELISA at B (n=48), D0 (n=46), D7 (n=48), D14 (n=48), D21(n=47) and D28 (n=33). The dotted line indicates the LLD for BPI ELISA(<100 pg/ml). Samples which fell below the LLD were assigned a value of50% the LLD. (C) Measurement of monocyte mCD14 and TLR4 surfaceexpression by flow cytometry revealed a nadir for mCD14 at D0 (n=10) andconcurrent peak TLR4 expression at D0 (n=9). Data represent meanfluorescence intensity (mCD14) or binding index (TLR4) geometricmeans+SEM of log transformed values labeled in original units. (D)Plasma IL-6 (n=37) and fever incidence (n=48) both peaked on D7. IL-6data represent geometric means+SEM of log transformed values labeled inoriginal units.

FIG. 2. BALB/c mice exhibit BM ablation and mortality at 7 Gy. (A) D30mortality after 6 (n=10), 6.5 (n=20) and 7 (n=20) Gy TBI differed bydose (p<0.001, Mantel-Cox log-rank). (B) Rapid onset of mucosal damagewas documented by peak colonic epithelial apoptosis at D3 (n=5)post-TBI, coincident with nadir in plasma citrulline levels (n=7),depicted normalized to levels before TBI (D0=100%). Data representmeans±SEM. *p<0.05 analyzed by 1-sample t test compared to D0; **p<0.001by Mann-Whitney. (C) Representative H&E stained femur sectiondemonstrates BM ablation D3 after 7 Gy TBI (4× magnification). (D) BMMNC counted after 0 Gy (normal controls, n=3/timepoint), 6.5 Gy(n=8/timepoint), and 7 Gy (n=8 on days 3 and 10, n=6 on D15 due togreater mortality). Data are the mean±SD of individual counts. Fewer BMMNC were present after 7 vs 6.5 Gy (D3 p=0.05, D10 p=0.0002, D15 p=0.02)Flow cytometry analysis of LK (E), and LSK cells (F) in BM of the samemice indicated 7 Gy produced prolonged reduction in progenitor and HSCnumbers. By D15, 6.5 mice had greater LK and LSK cell numbers than 7 Gymice (p=0.01 for both LK and LSK). Each symbol represents the absoluteLK or LSK number within BM from one limb of an individual animal. Medianvalues/group are indicated by horizontal bars. Hematologic data analyzedby Mann-Whitney.

FIG. 3. rBPI₂₁ in combination with ENR enhances survival of BALB/c miceafter 7 Gy TBI. (A) Survival of mice irradiated with 7 Gy given ENR plusrBPI₂₁ or VEH, ENR alone, or no treatment (denoted 7 Gy) 24 hours afterirradiation and continuing for 30 days. In a composite analysis of threereplicate experiments, survival of rBPI₂₁/ENR treated mice exceeded thatof the other groups (P<0.0001 by Mantel-Cox log rank, n=70 mice perarm). Survival of the rBPI_(L)/ENR group also exceed that of VEH/ENR,ENR, and 7 Gy (P<0.0001, 0.008, and <0.0001, respectively by pairwiseMantel-Cox log-rank). (B) Survival of mice irradiated with 7 Gy givenrBPI₂₁ or VEH (continued for either 14 or 30 days) plus ENR (continuedfor 30 days) or no treatment (denoted 7 Gy) 24 hours after irradiation.Survival was unaffected by duration of rBPI₂₁ treatment. Data wasanalyzed by pairwise Mantel Cox log-rank (n=20 mice per group).

FIG. 4. rBPI₂₁/ENR accelerates hematopoietic recovery after TBI-inducedaplasia. BALB/c BM MNC count (of one hind limb) and histopathology (fromthe contralateral hind limb) were assessed 10, 15, and 19 days aftervarious treatments. Data shown for (A) untreated, age-matched controls(normals) or (b) 7 Gy irradiated mice. Other mice received both 7 Gy TBIand the following treatments started 24 hrs after irradiation: (C) ENR,(D) VEH/ENR or (E) rBPI₂₁/ENR. Left panels: Each graph shows counts(mean±SD) of BM MNC flushed from a hind leg of 8 individual mice/groupexcept (B) where the high mortality (median survival 12-15 days)experienced by mice given 7 Gy alone resulted in n=2-8/timepoint. TherBPI₂₁/ENR combination resulted in improved BM cellularity as comparedto 7 Gy, ENR and VEH/ENR on D10 (p=0.0003, 0.001 and <0.0001,respectively), D15 (p=0.0007, p=0.001 and p=0.001, respectively) and D19(p=0.0006, p<0.0001 and p<0.0001, respectively) by Mann-Whitney. Dataare aggregated from two replicate studies. Similar results were obtainedin both studies. Right panels: Representative D19 H&E stained sectionsof the femurs of animals receiving indicated treatments demonstrate theclose correlation of BM MNC counts with BM histology.

FIG. 5. rBPI₂₁/ENR treatment results in restoration of BM cellularity tonormal levels by D30 after irradiation. BM histopathology (one hindlimb) and MNC count (contralateral hind limb) were assessed in micesurviving to D30. Representative femur histology is shown for (A)untreated, age-matched controls (normals) or (B) 7 Gy irradiated mice.Other mice received both 7 Gy TBI and the following treatments started24 hrs after irradiation: (C) ENR, (D) VEH/ENR or (E) rBPI₂₁/ENR. Inaddition to histology, corresponding counts of BM MNC flushed from ahind leg of individual mice were determined (F). Bars show mean±SD forn=4, 3, 12, 16, and 7 mice/group, respectively. The early, highmortality of 7 Gy alone and VEH/ENR treated mice limited the size ofthese cohorts. Only rBPI₂₁/ENR treatment resulted in BM MNC counts thatwere statistically indistinguishable from 0 Gy. rBPI₂₁/ENR MNC countsalso differed from counts in 7 Gy, ENR, and VEH/ENR (p=0.01, p=0.0002,p=0.001, respectively). Data from two replicate studies are shown.Similar results were obtained in all studies. Data analyzed byMann-Whitney.

FIG. 6. rBPI₂₁/ENR treatment is associated with more rapid expansion ofearly hematopoietic cells after 7 Gy TBI. Flow cytometry was used toquantify LK (left panels) and LSK (right panels) cells contained withinBM MNC of age-matched untreated controls (0 Gy) or mice administered 7Gy and initiated on no treatment (7 Gy), ENR, rBPI₂₁/ENR or VEH/ENRtreatments 24 hours thereafter. Results from D10, top panels, D19,middle panels, and D30, bottom panels, are shown. Box and whisker graphsdepict the range, 25^(th) and 75^(th) percentiles and median number ofLK or LSK phenotype cells within BM from one hind limb of each animal ineach treatment group. N=4 for 0 Gy controls at all timepoints. N=8mice/treatment on D10. N=6-8 mice/treatment at D19. Greater inequalityin survival to D30 resulted in n=3 (7 Gy), 12 (ENR), 16 (rBPI₂₁/ENR),and 7 (VEH/ENR) mice/group. Compared to 7 Gy, ENR or VEH/ENR, rBPI₂₁/ENRtreatment was associated with greater numbers of both LK and LSK cellsat the earlier time points (p=0.004 for all comparisons on D10 andp=0.004, 0.0003 and 0.0001 on D19, respectively). D30 LK and LSK contentof all groups, including controls, was equivalent. Data from tworeplicate experiments are shown. Similar results were obtained in allstudies. Data analyzed by Mann-Whitney.

FIG. 7. 7 Gy irradiation of BALB/c mice is associated with subsequentendotoxemia. Blood samples were obtained for endotoxin assay by LAL onthe days indicated and shown as mean±SEM. Endotoxin was present from D3onwards. N=9 mice/timepoint on days 0, 3, 12, n=6 on D6, and n=8 on D9.Mortality of 7 Gy alone treatment precluded evaluation of sufficientmice for analysis beyond D12.

FIG. 8. Injection site injury and inflammation result from BID injectionof rBPI₂₁ or VEH. During these radiation mitigation studies, some 7 Gyirradiated BALB/c mice received oral ENR only. Other 7 Gy irradiatedmice received ENR as well as twice daily injections of 250 μl of rBPI₂₁or its formulation buffer (denoted VEH) using sterile, single-use,insulin needles with fixed 28.5 G needles. Injections started 24 hoursafter radiation and continued until day 30. On either day 15 (B) or day19 (A, C), mice were humanely sacrificed and the underside of the dorsalskin was exposed for photo documentation of localized tissue injury.Images taken with a Nikon D90 digital camera.

FIG. 9. Trilineage hematopoiesis in cellular BM of rBPI₂₁/ENR treatedmice. BALB/c mice were irradiated to 7 GY and initiated rBPI₂₁/ENR 24hours thereafter. Mice were euthanized 19 days after irradiation. Lowpower images of H&E stained coronal sections of femur in rBPI₂₁/ENRtreated mouse were shown in FIG. 3. These images show higher powerimages at (A) 20× and (B) at 40×. BM demonstrates trilineagehematopoiesis without dysplasia, relative myeloid hyperplasia and robustrecovery of megakaryocytes.

FIG. 10. Gating strategy for determining LK and LSK cells in BM fromBALB/c mice by FACS. A gate is drawn on the FSC vs. SSC dot plot of BMcells in order to exclude small debris. Committed lineage cells weredetermined in the FL-4 channel (APC positive) vs. SSC, and gates drawnto bifurcate these from cells negative for lineage marker expression(neg-low APC fluorescence). Gating was confirmed through the use of amatched isotype control cocktail also conjugated to APC. Lineagenegative cells are visualized on Sca-1PE×c-kit-PerCP5.5 dualfluorescence dot plots to assess the content of Lin⁻Sca-1⁻c-kit⁺(LK—progenitor cells) and Lin⁻Sca-1⁺c-kit⁺ (LSK—stem cells) in mouse BM.Histograms shown are from the analysis of a normal mouse.

FIG. 11. Limited pilot clinical trial of rBPI₂₁ infusion in patientsundergoing allogeneic HSCT supports tolerability after myeloablativetherapy. Four of the six patients enrolled into the first cohort of anIRB-approved, multi-institutional Phase I-II pilot trial of rBPI₂₁administration in the setting of myeloablative HSCT received radiationbased treatment. All had hematologic malignancies, were aged 50-65 yrs(median 55), and signed consent. Subjects received cyclophosphamide and1360 (n=3) or 1400 (n=1) fractionated TBI for myeloablativeconditioning. (A) All patients received 4 mg/kg bolus IV rBPI₂₁ Day −1followed by continuous IV infusion of 6 mg/kg/day for 72 hours. Dose andduration of continuous infusion were to be escalated according to thedesign shown but the trial was discontinued by the Sponsor (XOMA (US)LLC) when the study lot of drug outdated. (B) Significant adverse eventsexperienced during the HSCT admission are shown.

FIG. 12. Effects of 14 and 30 days of rBPI₂₁ plus ENR on bone marrowmononuclear cells, L K and LK cells are equivalent. BALB/c mice wereirradiated to 7 Gy and treatments were initiated 24 hours thereafter.Some mice received twice daily subcutaneous rBPI₂₁ in combination withENR until D15 at which time the remainder were divided equally into onegroup called rBPI₂₁ (14)/ENR in which rBPI₂₁ was discontinued but ENRwas continued until D30, and another group, rBPI21 (30)/ENR, in whichboth rBPI₂₁ and ENR were continued until D30. The VEH/ENR group wastreated as previously described. rBPI₂₁ (14)/ENR and rBPI₂₁ (30)/ENR hadcomparable levels of bone marrow mononuclear cells (panels A, D), LK(panels B, E) and LSK (panels C, F) at all timepoints. Quantificationwas performed as per Methods. Bar graphs+SD depict bone marrowmononuclear cells, and box and whisker graphs depict the range, 25th and75th percentiles and median number of LK or LSK phenotype cells withinbone marrow from one hind limb of each animal in each treatment group.For the rBPI₂₁ (14)/ENR and rBPI₂₁ (30)/ENR groups. D15: n=4/group, D18:n=5-6/group, and D30: 8-10/group. Due to early mortality, there was n=1(7 Gy) and n=2 (VEH/ENR) at D18 and no survivors in those groups at D30.Values obtained from 2 normal animals are depicted as the 0 Gy values.Data obtained from a single study are shown.

FIG. 13. Peripheral blood counts are equivalent after 14 or 30 days ofrBPI₂₁ plus ENR treatment. Comparable levels of white blood cells (WBC),neutrophils, monocytes, platelets, and hemoglobin were measured in theperipheral blood of BALB/c mice treated with rBPI₂₁ for 14 days, rBPI₂₁(14)/ENR-red circles, as compared to treatment with rBPI₂₁ for 30 days,rBPI₂₁ (30)/ENR-gray circles. Treatments began 24 hours after 7 Gyirradiation. Peripheral blood counts were obtained as described inMethods. All mice received twice daily subcutaneous rBPI₂₁ incombination with ENR until D15 at which time 4 mice were bled forperipheral blood cell analysis. The remainder were divided equally intoone group called rBPI₂₁ (14)/ENR in which rBPI₂₁ was discontinued butENR was continued until D30, and another group, rBPI₂₁ (30)/ENR, inwhich both rBPI₂₁ and ENR were continued until D30. Results show themean+standard deviation of the peripheral blood count values measured onD18: n=5-6/group, and D30: 8-10/group. Values obtained from 2 normalanimals are depicted as the D0 values. Data obtained from a single studyare shown.

FIG. 14. (A-F) Granulocyte stimulating factor (G-CSF) levels areincreased in response to rBPI₂₁ treatment in irradiated and inunirradiated mice.

FIG. 15. (A-C) Murine keratinocyte chemoattractant (murine KC) levelsare increased in response to rBPI₂₁ treatment in in unirradiated mice.The stimulation of murine KC by rBPI₂₁ treatment is augmented by priorirradiation.

FIG. 16. Monocyte chemotactic protein-1 (MCP-1), also known as Chemokine(C—C motif) ligand 2 (CCL2) levels are increased by rBPI₂₁ treatment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention, in one aspect, relates to the surprisingdiscovery that bactericidal/permeability increasing protein (BPI) and/orits congeners, alone or in combination with an antibiotic, can mitigatetissue injury resulting from exposure to radiation, chemotherapy, ordisease. As described herein, BPI and/or its congeners, alone or incombination with an antibiotic, can be used to mitigate hematopoietictoxicity, stimulate hematologic function and aid bone marrow recovery insubjects (individuals) exposed to accidental or incidental radiation,and/or in subjects with severe myeloblation, involving depletion/failureof bone marrow cells.

As used herein, a subject in need of BPI and/or its congeners therapy,is a subject having a decrease (partial or complete) in bone marrowfunction. In some embodiments, the subject has insufficienthematopoiesis in one or more blood cell types or blood lineages. In someembodiments, the subject is exposed to radiation, chemoradiotherapy or atoxin, or has a disease, or a drug or biologic-mediated hematopoieticinjury that results in a decrease (partial or complete) in bone marrowfunction in the subject. Examples of diseases that cause a decrease(partial or complete) in hone marrow function include, but are notlimited to acute and chronic inflammation, infection, aplastic anemia,Fanconi anemia, Bloom syndrome, reticular dysgenesis, Kostmann syndrome,congenital benign neutropenia, neonatal sepsis, myelodysplasticsyndrome, Diamond-Blackfan anemia and congenital or acquired marrowfailure syndrome. The subject is not otherwise in need of treatment withBPI and/or its congeners. In some such embodiments, the subject does nothave an infectious disease.

In some embodiments, the subject is exposed to levels of radiationsufficient to cause unwanted tissue injury and/or hematopoietictoxicity. In some embodiments, the subject has been exposed to-radiationsufficient to cause tissue injury and/or hematopoietic toxicity prior toBPI and/or its congeners therapy. In some embodiments, the subject hasnot been exposed to radiation and prior to exposure radiation sufficientto cause tissue injury and/or hematopoietic toxicity receives treatmentwith BPI and/or its congeners in anticipation of future radiationexposure. In some embodiments, the subject is being exposed to radiationduring BPI and/or its congeners therapy. Radiation exposure includes,but is not limited to, accidental exposure, exposure resulting from anuclear attack, and medical radiation therapy such as local therapy andlow and high dose total body irradiation. In some embodiments, thesubject has cancer, and has undergone, is undergoing or will undergoradiation therapy, chemoradiotherapy and/or chemotherapy.

According to some aspects of the invention, a method for mitigatingradiation-induced tissue injury of any type is provided. Exposure toradiation is toxic at low doses and life threatening at high doses. Thetissues which are most vulnerable to radiation-induced damage includethe hematopoietic system and the gastrointestinal tract (GI). Moderatedoses of radiation can cause a rapid reduction in blood cells counts,including loss of circulating lymphocytes and a reduction in mitoticallyactive hematopoietic progenitor cells. Reduction in blood cell count isassociated, among other things, with increased risk of infections, andcancer development. Higher doses of radiation can lead to more severeand often permanent bone marrow damage, resulting from loss of bonemarrow stem cell populations. Thus, the tissue injury may be, forexample, a decrease in blood cell count, loss of bone marrow stem cellpopulations, or hematopoietic toxicity.

According to some aspects of the invention, a method for mitigatinghematopoietic toxicity is provided. The method comprises administeringBPI and/or its congeners, alone or in combination with an antibiotic.The term ‘hematopoietic toxicity’ refers to a toxicity thatsubstantially arises from exposure to radiation that adversely affectsthe hematopoietic system of an individual (subject). Alternatively,hematopoietic toxicity may result from exposure of the subject to atoxin or a disease or a genetic predisposition to hematopoietic injury.This adverse effect can be manifested in the subject broadly, in thatthe levels of many hematopoietic cell types are altered (differ fromlevels considered to be normal), as a result of the radiation exposure,chemotherapy, toxin or disease, or the adverse effect can be manifestedin the subject more specifically, in that only one or a fewhematopoietic cell types differ from levels considered to be normal as aresult of the exposure to the radiation, chemotherapy, toxin or disease.

BPI and/or its congeners and antibiotic(s) are administered formitigating tissue injury, such as hematopoietic toxicity. As usedherein, the term “mitigate” refers to a reduction in the extent ofdisease, chemotherapy, toxin or radiation-induced tissue damage (damageis less than would occur in the absence of BPI/congener treatment). Assuch, the reduction in the extent of disease, chemotherapy, toxin orradiation-induced tissue damage may be evaluated in terms of animprovement in the health of the tissue in treated subjects. Animprovement in the health of tissues of treated subjects may bedetermined by examining the health of the tissue in treated subjectsversus the health of tissue in control subjects (subjects receiving thesame amount of radiation exposure treated subjects but not receiving theBPI therapy). The health of the tissue may be measured by any variety ofmethods known to those of ordinary skill in the art, including directand indirect measurements. Direct measurements are those such asmeasuring cell count. In some embodiments, the tissue injury measuredmay be necrosis of the tissue, and/or a decrease in blood cell count. Insome embodiments, the improvement in the health of the tissue may bemeasured by evaluating the function of the hematopoietic system usingend points such as hematocrit, white blood cell count, incorporation oftritiated thymidine into bone marrow DNA, spleen weight, number ofburst-forming units-erythroid or number of colony forming units(erythroid, granulocyte, macrophage and megakaryocyte forming lineages)from spleen or hone marrow obtained from humerus or femur or enumerationof circulating hematopoietic stem cells or other primitive hematopoieticcells in the peripheral circulation.

According to some aspects of the invention, a method for bone marrowrecovery is provided. The method comprises administering BPI and/or itscongeners, alone or in combination with an antibiotic. “Bone marrowrecovery” means the process whereby hone marrow that has been damaged byradiation, chemotherapy, disease or toxins is restored to its normal ornear normal state (function), or where a measurable improvement in bonemarrow function is obtained. Bone marrow function is the process wherebythe various blood cell types or lineages are produced from thehematopoietic (blood) stem cells. The end points that can be used tomeasure bone marrow recovery include, but are not limited to hematocrit,white blood cell count, incorporation of tritiated thymidine into honemarrow DNA, spleen weight, number of burst-forming units-erythroid ornumber of colony forming units (erythroid, granulocyte, macrophage andmegakaryocyte forming lineages) from spleen or bone marrow obtained fromhumerus or femur or enumeration of circulating hematopoietic stem cellsor other primitive hematopoietic cells in the peripheral circulation. Insome embodiments, the subject is exposed to radiation or a toxin, or hasa disease, or a drug or biologic-mediated hematopoietic injury thatresults in a decrease (partial or complete) in bone marrow function inthe subject. Examples of diseases that cause a decrease (partial orcomplete) in bone marrow function include, but are not limited to acuteand chronic inflammation, infection, aplastic anemia, Fanconi syndrome,Bloom syndrome, reticular dysgenesis, Kostmann syndrome, congenitalbenign neutopenia, neonatal sepsis, myelodysplastic syndrome,Diamond-Blackfan anemia and congenital or acquired marrow failuresyndrome.

According to some aspects of the invention, a method for stimulatinghematopoiesis is provided. The method comprises administering BPI and/orits congeners, alone or in combination with an antibiotic. “Stimulationof hematopoiesis” generally refers to an increase in one or morehematopoietic cell types or lineages, and especially relates to astimulation or enhancement of one or more hematopoietic cell types orlineages in cases where a subject has a deficiency in one or morehematopoietic cell types or lineages. The deficiency in one or morehematopoietic cell types or lineages may be caused by exposure toradiation or a toxin, a disease, drug or biologic-mediated hematopoieticinjury. Examples of diseases that cause a deficiency in one or morehematopoietic cell types or lineages include, but are not limited toacute and chronic inflammation, infection, aplastic anemia, fanconisyndrome, Bloom syndrome, reticular dysgenesis, Kostmann syndrome,congenital benign neutopenia, neonatal sepsis, myelodysplastic syndrome,Diamond-Blackfan anemia and congenital or acquired marrow failuresyndrome. Hematopoietic deficiency may comprise lymphopenia, leukopenia,neutropenia, erythropenia, megakaryocytopenia, a deficiency inplatelets, a deficiency in monocytes, a deficiency in lymphocyctes, adeficiency in erythrocytes, deficiency in neutrophils, a deficiency in Tcells, or B cells specifically, a deficiency in granulocytes, and/or adeficiency in dendritic cells.

The compounds useful in the methods of the invention are BPI, itsbiologically active fragments, analogs, variants and/or its congeners.BPI, a 50-55 kDa cationic antimicrobial protein found primarily in theazurophilic granules of human polymorphonuclear neutrophils, has thehighest affinity (pM-nM) for a variety of bacteria-associated andcell-free forms of endotoxin. BPI binding to endotoxin promotes killingand clearance of Gram-negative bacteria and inhibits endotoxin-inducedinflammation and apoptosis by precluding endotoxin binding to thecellular pro-inflammatory endotoxin receptor complex composed of mCD14,MD-2, and TLR4. Most BPI is intracellular but plasma levels of BPI risewith neutrophil activation and degranulation. Stable BPI congenersinclude, but are not limited to rBPI₂₁, rBPI₂₃, rBPI₅₀,rBPI(10-193)ala¹³² and a N-terminal fragment of BPI having a molecularweight approximately between 20 to 25 kD. Preparation of BPI and itscongeners has been have been described in the art in publications suchas U.S. Pat. No. 6,268,345, U.S. Pat. No. 6,599,880, U.S. Pat. No.5,420,019, U.S. Pat. No. 5,980,897 and US Pub No. 2008/0031874.

BPI and/or its congeners may be given in combination with an antibiotic(at least one, one or more antibiotic). In some embodiments, theantibiotic is a quinolone. In some embodiments, the quinolone is afluoroquinolone, which has a fluorine atom attached to the central ringsystem, typically at position 6 or 7. Examples of quinolone antibioticsadministered in combination with BPI and/or its congeners include, butare not limited to, moxifloxacin, ciprofloxacin, levofloxacin,garenoxacin, and delafloxacin.

BPI and/or its congeners and the antibiotic(s) may be administeredsimultaneously or sequentially. When BPI and/or its congeners and theantibiotic(s) are administered simultaneously, they can be administeredin the same or separate formulation(s), and are administered atsubstantially the same time. The administration of the antibiotic(s) andBPI and/or its congeners may also be sequential; the two need only beadministered sufficiently close in time to have the desired effect onbone marrow function. In certain embodiments, the antibiotic(s) areadministered before BPI and/or its congeners or after the administrationof BPI and/or its congeners. The separation in time between theadministration of these compounds may be a matter of minutes, 5 hours,12 hours, 24 hours, 48 hours, or 96 hours, or it may be longer.

The compounds of the present invention are administered in effectiveamounts. An effective amount is a dose sufficient to provide a medicallydesirable result and can be determined by one of skill in the art usingroutine methods. In the treatment of radiation-induced tissue damage, aneffective amount will be that amount necessary to inhibit (partially orcompletely) tissue damage caused by exposure to radiation. In someembodiments, an effective amount is an amount which results inimprovement in the condition being treated. In some embodiments, aneffective amount may depend on the type and extent of radiationexposure, and/or the use of one or more additional therapeutic agents.However, one of skill in the art can determine appropriate doses andranges of BPI/congener and antibiotic(s) to use, for example based on invitro and/or in vivo testing and/or other knowledge of compound dosages.It should be appreciated that in some embodiments, BPI and/or itscongeners and antibiotic(s) described herein may be administered indosages that inhibit radiation-caused injury of non-cancerous tissuesand cells, without materially interfering with the killing of canceroustissues and cells.

When administered to a subject, effective amounts of BPI/congener andantibiotic(s) will depend on, for example, the severity of the injury;individual patient parameters including age, physical condition, sizeand weight, concurrent treatment, frequency of treatment, and the modeof administration. These factors are well known to those of ordinaryskill in the art and can be addressed with no more than routineexperimentation. In some embodiments, a maximum dose is used, that is,the highest safe dose according to sound medical judgment.

An effective amount typically will vary from about 0.001 mg/kg to about1000 mg/kg, from about 0.01 mg/kg to about 750 mg/kg, from about 0.1mg/kg to about 500 mg/kg, from about 1.0 mg/kg to about 250 mg/kg, fromabout 10.0 mg/kg to about 150 mg/kg in one or more dose administrations,for one or several or many days (depending on the mode of administrationand the factors discussed above).

Actual dosage levels of the BPI/congener and antibiotic(s) can be variedto obtain an amount that is effective to achieve the desired therapeuticresponse for a particular patient, compositions, and mode ofadministration. The selected dosage level depends upon the activity ofthe particular compound, the route of administration, the severity ofthe radiation exposure, and prior medical history of the patient beingtreated. However, it is within the skill of the art to start treatmentwith doses of the compound at levels lower than required to achieve thedesired therapeutic effort and to gradually increase the dosage untilthe desired effect is achieved.

BPI and/or its congeners and antibiotic(s) may be administered any timeonce the subject is diagnosed as having a decrease (partial or complete)in bone marrow function. In some embodiments, BPI and/or its congenersand antibiotic(s) is administered any time after the subject isdiagnosed as having a decreased blood cell count as compared to expectednormal levels. In some embodiments, BPI and/or its congeners andantibiotic(s) are administered before, during or after exposure of thesubject to levels of radiation causing tissue damage. In someembodiments, BPI and/or its congeners and antibiotic(s) are administeredbefore radiation exposure, but close enough in time to the radiationexposure to inhibit radiation-induced tissue damage. In someembodiments, BPI and/or its congeners and antibiotic(s) are administeredany time up to 1 day before the radiation exposure. In some embodiments,BPI and/or its congeners and antibiotic(s) are administered between 1and 24 hours before radiation exposure. In some embodiments, BPI and/orits congeners and antibiotic(s) are administered within 12 hours ofradiation exposure. BPI and/or its congeners and antibiotic(s) may alsobe administered during radiation exposure. In some embodiments, BPIand/or its congeners and antibiotic(s) are administered after radiationexposure, yet close enough in time to the radiation exposure to have thedesired effect of protecting tissue from radiation-induced tissueinjury. In some embodiments, BPI and/or its congeners and antibiotic(s)is administered any time up to 3 days post-exposure. In someembodiments, BPI and/or its congeners and antibiotic(s) are administeredbetween 1-60 hours following radiation exposure. In some embodiments,BPI and/or its congeners and antibiotic(s) are administered within 24 or48 hours of radiation exposure. In some embodiments, the subject hascancer and the BPI and/or its congeners and antibiotic(s) areadministered at least 1 hour, at least 12 hours, at least 24 hours, orat least 48 hours following radiation therapy, chemoradiotherapy, orchemotherapy, but not more than 72 hours following radiation therapy,chemoradiotherapy, or chemotherapy.

BPI and/or its congeners and antibiotic(s) and pharmaceuticalcompositions containing BPI and/or its congeners and antibiotic(s) areadministered to a subject by any suitable route. For example, thecompositions can be administered orally, including sublingually,rectally, parenterally, intracistemally, intravaginally,intraperitoneally, topically and transdermally (as by powders,ointments, or drops), bucally, or nasally. The term “parenteral”administration as used herein refers to modes of administration otherthan through the gastrointestinal tract, which include intravenous,intramuscular, intraperitoneal, intrasternal, intramammary, intraocular,retrobulbar, intrapulmonary, intrathecal, subcutaneous andintraarticular injection and infusion. Surgical implantation also iscontemplated, including, for example, embedding a composition of theinvention in the body such as, for example, in the brain. In someembodiments, the compositions may be administered systemically.

Pharmaceutical compositions of the invention for parenteral injectioncomprise pharmaceutically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions, or emulsions, as well as sterilepowders for reconstitution into sterile injectable solutions ordispersions just prior to use. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents, or vehicles include waterethanol, polyols (such as, glycerol, propylene glycol, polyethyleneglycol, and the like), and suitable mixtures thereof, vegetable oils(such, as olive oil), and injectable organic esters such as ethyloleate. Proper fluidity can be maintained, for example, by the use ofcoating materials such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions, and by the use of surfactants.

These compositions also can contain preservatives, wetting agents,emulsifying agents, and dispersing agents. Prevention of the action ofmicroorganisms can be ensured by the inclusion of various antibacterialand antifungal agents, for example, paraben, chlorobutanol, phenolsorbic acid, and the like. It also may be desirable to include isotonicagents such as sugars, sodium chloride, and the like. Prolongedabsorption of the injectable pharmaceutical form can be brought about bythe inclusion of agents which delay absorption, such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of the drug, it isdesirable to slow the absorption of the drug from a subcutaneous orintramuscular injection. This result can be accomplished by the use of aliquid suspension of amorphous materials with poor water solubility.Delayed absorption of a parenterally administered drug also isaccomplished by dissolving or suspending the drug in an oil vehicle.Likewise, injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such apolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations also are prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

The invention provides methods for oral administration of apharmaceutical composition of the invention. Oral solid dosage forms aredescribed generally in Remington's Pharmaceutical Sciences, 18th Ed.,1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89. Solid dosageforms for oral administration include capsules, tablets, pills, powders,troches or lozenges, cachets, pellets, and granules. Also, liposomal orproteinoid encapsulation can be used to formulate the presentcompositions (as, for example, proteinoid microspheres reported in U.S.Pat. No. 4,925,673). Liposomal encapsulation may include liposomes thatare derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556).

In such solid dosage forms, the active compound is mixed with, orchemically modified to include, at least one inert, pharmaceuticallyacceptable excipient or carrier. The excipient or carrier may permitincreased uptake of the compound, overall stability of the compoundand/or circulation time of the compound in the body. Excipients andcarriers include, for example, sodium citrate or dicalcium phosphateand/or (a) fillers or extenders such as starches, lactose, sucrose,glucose, cellulose, modified dextrans, mannitol, and silicic acid, aswell as inorganic salts such as calcium triphosphate, magnesiumcarbonate and sodium chloride, and commercially available diluents suchas FAST-FLO®, EMDEX®, STA-RX 1500®, EMCOMPRESS® and AVICEL®, (b) binderssuch as, for example, methylcellulose ethylcellulose,hydroxypropyhnethyl cellulose, carboxymethylcellulose, gums (e.g.,alginates, acacia), gelatin, polyvinylpyrrolidone, and sucrose, (c)humectants, such as glycerol, (d) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, sodium carbonate, starch including the commercialdisintegrant based on starch, EXPLOTAB®, sodium starch glycolate,AMBERLITE®, sodium carboxymethylcellulose, ultramylopectin, gelatin,orange peel, carboxymethyl cellulose, natural sponge, bentonite,insoluble cationic exchange resins, and powdered gums such as agar,karaya or tragacanth; (e) solution retarding agents such a paraffin, (f)absorption accelerators, such as quaternary ammonium compounds and fattyacids including oleic acid, linoleic acid, and linolenic acid (g)wetting agents, such as, for example, cetyl alcohol and glycerolmonosterate, anionic detergent surfactants including sodium laurylsulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate,cationic detergents, such as benzalkonium chloride or benzethoniumchloride, nonionic detergents including lauromacrogol 400, polyoxyl 40stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60,glycerol monostearate, polysorbate 40, 60, 65, and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose; (h)absorbents, such as kaolin and bentonite clay, (i) lubricants, such astalc, calcium sterate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, polytetrafluoroethylene (PTFE), liquid paraffin,vegetable oils, waxes, CARBOWAX® 4000, CARBOWAX® 6000, magnesium laurylsulfate, and mixtures thereof; (j) glidants that improve the flowproperties of the drug during formulation and aid rearrangement duringcompression that include starch, talc, pyrogenic silica, and hydratedsilicoaluminate. In the case of capsules, tablets, and pills, the dosageform also can comprise buffering agents.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. Liquid dosage forms for oral administration includepharmaceutically acceptable emulsions, solutions, suspensions, syrups,and elixirs. In addition to the active compounds, the liquid dosageforms can contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol ethyl carbonate ethyl acetate,benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethyl formamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydroflirfurylalcohol, polyethylene glycols, fatty acid esters of sorbitan, andmixtures thereof.

Also contemplated herein is pulmonary delivery of the compounds of theinvention. The compound is delivered to the lungs of a mammal whileinhaling. Contemplated for use in the practice of this invention are awide range of mechanical devices designed for pulmonary delivery oftherapeutic products, including, but not limited to, nebulizers, metereddose inhalers, and powder inhalers, all of which are familiar to thoseskilled in the art. All such devices require the use of formulationssuitable for the dispensing of a compound of the invention. Typically,each formulation is specific to the type of device employed and caninvolve the use of an appropriate propellant material, in addition todiluents, adjuvants, and/or carriers useful in therapy.

The present invention is further illustrated by the following Example,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES

Materials and Methods

Patient Characteristics

Patients (n=48) undergoing myeloablative allogeneic HSCT from 2005-2009at Children's Hospital Boston (CHB) or Brigham and Women's Hospital(BWH) were recruited prospectively on an Institutional Review Boardapproved study. All participants and/or legal guardians gave consentand/or assent as appropriate. Age ranged from 1-60 years. Myeloablativeregimens for hematologic malignancies (n=46) were chemoradiotherapy withTBI, 1400 cGy (n=38) or 1375 cGy (n=1) or combination chemotherapyincluding busulfan>14 mg/kg oral or IV equivalent (n=7). Myeloablationfor aplastic anemia was cyclophosphamide 200 mg/kg (6 gm/M²) plus ATG(n=2). Sixteen patients received BM and 32 received PB stem cells.Supportive care was per institutional routine. (48, 49). Prophylacticoral nonabsorbable antibiotics were administered: bacitracin and eitherpolymyxin (BWH) or vancomycin (CHB). Blood counts and cultures wereperformed per routine in clinical laboratories. Sixteen patients hadbacteremia with either Gram-positive (n=15) or Gram-negative (n=1)organisms. Temperature was recorded using the maximal value within ±1day of sample acquisition. Endotoxin activity assay (EAA) measurementswere added when the EAA became available.

Blood Collection and Plasma Preparation

PB samples were drawn into K2-EDTA or sodium heparin Vacutainers™,(Becton-Dickinson (BD), Franklin Lakes, N.J.) before conditioning(Baseline, B), on the day of HSCT (D0), and weekly ±1 day. PB was spun1200 g for 5 min at 4° C., recovered, and stored in aliquots inpyrogen-free tubes at −80° C.

Human BPI ELISA

BPI was measured by ELISA (HyCult, Uden, The Netherlands), according tothe manufacturers' instructions.

Endotoxin Measurement in Human PB

Endotoxin was measured by EAA according to the manufacturer'sinstructions (Spectral Diagnostics, Toronto, Canada). (27)

Human IL-6 ELISA

IL-6 was measured by flow cytometry (Mono, DakoCytomation, Glostrup,Denmark) using antibody coated fluorescent beads (Cytometric Bead ArrayBD Flex Sets, BD BioSciences, San Jose, Calif.) and Summit v4.0 software(DakoCytomation).

Measurement of mCD14 and TLR4

Monocyte surface expression of CD14 and TLR4 was measured withantigen-specific or isotype control monoclonal antibodies (eBioSciences,San Diego, Calif.) as previously described. (50)

In Vivo Radiation Mitigation Studies with rBPI₂₁ and Enrofloxacin

Male BALB/c mice (Stock #028, Charles River, Wilmington, Mass.) wereacclimated prior to irradiation at age 12 weeks. Studies were conductedin accordance with Dana-Farber Cancer Institute's ACUC-approved policiesand protocols. Mice were placed into a Rad Disk™ rodent microisolationirradiation cage (Braintree Scientific, Braintree, Mass.) andadministered a single 7 Gy dose by a Gammacell® 40Exactor (BestTheratronics, Ottawa, Ontario) cesium source irradiator. Twenty-fourhours thereafter, mice were either left untreated (7 Gy), or receivedone or more of the following treatments for 30 days: 1) rBPI₂₁, (XOMA(US) LLC, Berkeley, Calif.), 250 μl per injection of a 2 mg/ml stockconstituted in formulation buffer and administered SC twice daily, 6-8hours apart (rBPI₂₁/mouse was ˜42 mg/kg/day); 2) 250 μl of the rBPI₂₁formulation buffer (denoted VEH) consisting of 0.33 g/L citric acid,1.01 g/L sodium citrate, 8.76 g/L sodium chloride, 2.0 g/L PoloxamerP188, and 2.0 g/L polysorbate 80, (all Sigma, St. Louis, Mo.) dissolvedin water for injection, pH adjusted to 5.0, and filter sterilized; 3)Baytril® (enrofloxacin, MedVets, Sandy, Utah) at 10 mg/kg/day by oralgavage via 25 G feeding needles (Cadence Science, Cranston, R.I.) forthe first 5-7 days, after which mice continued to receive antibiotic adlib in water bottles until study termination or death. All mice wereobserved at least twice daily. Moribund mice were euthanized via CO,asphyxiation. At scheduled time points, mice were sacrificed humanelyvia isoflurane anesthetic overdose (IsoFlo®-Abbott Labs, Abbott Park,Ill.).

Blood and Tissue Preparation

CBCs were performed on a Hemavet 950 FS hematology analyzer (DrewScientific, Waterbury, Conn.) with EDTA-anticoagulated(Becton-Dickinson, Franklin Lakes, N.J.) cardiac blood. Plasma wasobtained by mixing blood with pyrogen-free heparin (APP Pharmaceutical,Schaumburg, Ill.), in pyrogen-free Eppendorf tubes (USA Scientific) andcentrifugation at 14,000 rpm for 10 minutes. Single use aliquots werestored at −80° C. In some studies, femurs and tibiae from one leg/animalwere dissected, fixed for 24 hours in 10% neutral buffered formalin(Fisher Scientific, Pittsburgh, Pa.), and processed, including coronalsectioning and hematoxylin and eosin (H&E) staining (SpecializedHistopathology Services-Longwood, Boston, Mass.). Contralateral femursand tibiae were taken for BM MNC enumeration and flow cytometry byflushing cells from the bones with cold RPMI 1640 medium supplementedwith 10% FBS (JRH Biosciences, Lenexa, Kans.), L-glutamine, HEPES,pen/strep and gentamicin (all from Invitrogen, Carlsbad, Calif.). Redblood cells were lysed with hypotonic lysing buffer (Sigma). BM MNC wereenumerated by trypan blue staining; viability was typically >90-95%.

Citrulline Determinations

Samples were analyzed with the MassTrak Amino Acid Analysis (AAA) system(Waters, Milford Mass. USA) with AccQTag™ derivatization andultraviolet/visible detection.

Histopathologic Evaluations

A Board-certified hematopathologist (JK) assessed femoral BM cellularityon decalcified, formalin fixed, H&E stained paraffin-fixed sectionsusing an Olympus BX51 microscope and an Olympus DP71 camera with DPCapture software. For each animal, 2 slides with 2 fields/slide werescored for the percent of BM space occupied by hematopoietic cells. ABoard-certified pathologist (J-AV) enumerated apoptotic bodies/400×field in triplicate samples of H&E-stained paraffin-fixed colonsections. Samples from normal mice were identified, but all others weredeidentified and presented in random order for analysis.

Endotoxin Measurement in Murine Plasma

Endotoxin was measured using the Limulus amoebocyte lysate (LAL) assayaccording to the manufacturer's instructions (Charles River, Boston,Mass.), and as previously described. (51)

BM FACS Analysis

BM cells were preincubated with 2% Rat anti-mouse CD16/CD32 and 1%normal rat serum for Fc blocking prior to staining cells bearinghematopoietic lineage markers (CD3ε, CD45/B220, CD11b, Ly-6G/Ly-6C, TER119) with a cocktail of APC-conjugated lineage specific antibodies, orequivalent concentration of APC conjugated isotype controlimmunoglobins, 1:20 dilutions of PE-rat anti-mouse Sca 1 (clone D7) andPerCP-Cy5.5-rat anti-mouse c-Kit (clone 2B8), all from BD. Cells werestained for 25 minutes at 4° C., washed 2× with cold DPBS, andresuspended in 0.4% paraformaldehyde. 100,000 events were acquired on aFACScalibur™ flow cytometer (BD) and analyzed with FlowJo v.7.0.5(Treestar) software. Cells negative for lineage marker expression wereassessed for percentages of lin⁻Sca-1⁻c-kit⁺ (LK) and lin⁻Sca-1⁺c-kit⁺(LSK) in BM (FIG. 10, gating strategy). Data from normal mice wereconsistent with published reports in naïve BALB/c mice. (52)

Statistics

For the human HSCT study, samples with undetectable analytes wereassigned a value at half the lower limit of detection. For ANC, PLT,BPI, TLR4 and IL-6, data were analyzed after logarithmic transformation,as this yielded distributions that were more approximately normal. Forthese data, geometric means and error bars indicating +1 standard errorof the mean (SEM) of the log values were then transformed back tooriginal units and plotted on a logarithmic axis. The Wilcoxon signedrank test for matched pairs was employed when comparing values for thesame patients at different time points, with values compared tobaseline. Comparisons between subjects with or without fever wereevaluated using the Mann-Whitney test. When assessing correlationsbetween different parameters, within-subject correlations werecalculated using the Spearman correlation coefficient and data frommultiple time-points. The calculated coefficients were averaged over thedifferent subjects and significance tested with the signed rank test.Unless otherwise noted, all p-values were two-sided. Statisticalsignificance and graphic output were generated using Prism v. 4.0a(GraphPad Software; San Diego, Calif.) and SAS v. 9.1 (SAS Institute,Cary, N.C.). Statistical analysis for murine experiments was performedwith Graph Pad Prism Version 5. Mantel-Cox log-rank was used to comparesurvival curves. Two tailed t tests (Mann-Whitney) are performedthroughout except for citrulline data in which the data were analyzed by1-sample t test as compared to theoretical mean of 100% and forhematologic analyses (Table 1) in which unpaired t tests were performed.Unpaired t tests do not assume equal variance. In all experiments, a Pvalue of <0.05 was used to reject the null hypothesis. Where indicatedin figures, *p<0.05, **p<0.01, ***p<0.001.

TABLE I Peripheral blood counts after TBI by treatment group. WBCNeutrophils Monocytes Hb PLT (K/μL) (K/μL) (K/μL) (g/dL) (K/μL) 0 Gy4.08 0.84 0.24 15.7 914  (2.14-11.48) (0.46-2.4)  (0.08-0.37)(14.6-17.8)  (754-2015) 7 Gy D15 0.36 ▴ 0.06 ▴ 0.025 ▴ 10.4 ▴ 195 ▴(0.22-0.38) (0.03-0.08) (0.01-0.04)  (8.8-11.3)  (95-225) D19 0.46 ▴0.115 0.05 ▴ 7.7 ▴ 280.5 ▴ (0.32-0.61) (0.11-0.12) (0.04-0.06) (7.5-7.9)(224-337) D30 3.98 1.66 ▴ 0.66 ▴ 13.4 ▴ 692 (2.64-9.68) (1.16-4.86)(0.54-1.58) (13.1-14.3) (680-956) ENR/BPI D15 0.3 ▴ 0.07 ▴ ▾ 0.01 ▴ 7.8▴ ● 229 ▴ (0.20-0.32) (0.06-0.08)   (0-0.02) (6.7-9.4)  (89-254) D192.63 ▪ ▾ 0.73 ▪▾ 0.26 ▪▾ 10.6 ▴ ▪ 599.5 ▴ ▪ ▾ (1.42-7.32) (0.29-1.27)(0.16-0.62)  (7.5-11.7) (360-785) D30 3.62 ▾ 1.69 ▴ ▾ 0.44 ▴ 13.7 ▴ 849(0.72-5.5)  (0.15-3.94) (0.07-1.75) (11.0-14.3)  (407-1015) ENR/VEH D150.22 ▴ 0.005 ▴ 0.005 ▴ 7.1 ▴ ● ▾ 127 ▴ (0.14-0.40)   (0-0.09)   (0-0.03)(2.4-8.0)  (61-231) D19 0.38 ▴ 0.07 ▴ ▾ 0.02 ▴ ▾ 4.9 ▴ ● ▾ 199 ▴(0.28-0.74) (0.02-0.18) (0.01-0.04) (2.4-7.4)  (95-261) D30 2.3 1.05 0.413.7▴ 753 (1.42-12.5)  (0.4-6.31) (0.19-3.02) (12.8-14.1) (471-1175) ENRD15 0.24 ▴ 0 ▴ ● 0 ▴ ● 8.80 ▴ ▪ 196 ▴ (0.14-0.30)  (5.6-10.9) (113-277)D19 0.21 ▴ 0 ▴ ● ▪ 0 ▴ ● ▪ 8.35 ▴ ▪ 250.5 ▴ (0.18-0.32)  (6.7-10.9)(185-318) D30 1.84 ▴ ● 0.55 ● 0.14 ● 13.1 ▴ 713.5 ▴ (0.36-4.8) 0.06-2.34 (0.03-1.03)  (2.9-15.5) (128-923)The following blood counts in Table I are values where the median fallsinto the normal range of the age-matched 0 Gy controls: for WBC: 7 Gy atD30, ENR/BPI at D19, ENR/BPI at D30, and ENR/VEH at D30; forNeutrophils: 7 Gy at D30, ENR/BPI at D19, ENR/BPI at D30, ENR/VEH atD30, and ENR at D30; for Monocytes: 7 Gy at D30, ENR/BPI at D19, ENR/BPIat D30, ENR/VEH at D30, and ENR at D30; for PLT: ENR/BPI at D30. ▴statistically significant versus 0 Gy; ● statistically significantversus 7 Gy; ▪ statistically significant versus VEH/ENR; ▾ statisticallysignificant versus ENR. All p<0.05.ResultsHuman Myeloablative HSCT is Associated with Early Neutropenia,Endotoxemia, Deficiency of BPI and Evidence of Host Responses toEndotoxin

We examined endotoxin and BPI plasma levels in patients who underwentmyeloablative conditioning for HSCT. Thirty-nine of 48 patients receivedchemoradiotherapy including 1375 (n=1) or 1400 (n=38) cGy TBI while 9received ablative combination chemotherapy alone. As expected,myeloablative therapy followed by allogeneic HSC infusion resulted in afall and recovery in PB counts (FIG. 1A). By the completion ofmyeloablative conditioning (D0), endotoxemia was readily detectable(FIG. 1B). Simultaneously, plasma BPI concentrations declined rapidly(D7 median decrease 71-fold, inter-quartile range 9-193-fold; FIG. 1B),correlating with the absolute neutrophil count (ANC; Spearman r=0.66;p<0.001). At the ANC nadir (D7), plasma BPI was undetectable (<100pg/mL) in 37/48 patients (77%) and 80% of patients evaluated byendotoxin activity assay (27) were endotoxemic.

The TLR4 and mCD14 components of the TLR endotoxin receptor onperipheral blood (PB) monocytes exhibited increased and decreasedsurface expression, respectively, consistent with early PB mononuclearcell exposure to bioactive endotoxin (28, 29) (FIG. 1C). Subsequentelevation of IL-6 and fever, well-described downstream sequelae of TLR4engagement by endotoxin, were maximal at the BPI nadir (D7, FIG. 1D).Intrapatient changes in IL-6 concentrations were positively correlatedwith the EAA (Spearman 0.48, p=0.01), and higher IL-6 levelsconcentrations were inversely correlated with BPI levels (Spearman−0.30, p<0.0001). Fever and BPI levels showed no association on D7,perhaps because nearly 80% of patients had undetectable BPI. However, onD14 patients with fever had lower BPI levels than afebrile patients(medians: undetectable vs. 3475 pg/mL, p=0.01). Notably, lower plasmaBPI concentrations on D0, immediately prior to HSC infusion, wereassociated with longer time to neutrophil engraftment (p=0.03, Spearmanr=−0.32), and with a trend towards longer time to platelet recovery(Spearman r=−0.26, p=0.08).

These findings suggest that BPI deficiency coupled with endotoxemiacould contribute to endotoxin-related toxicity after myeloablation andraise the possibility that BPI supplementation might attenuate thesetoxicities. Whereas administration of HSC enables survival aftermyeloablation, HSC support would not be feasible after unintendedradiation exposure. As radiation mitigation without HSC cannot beaddressed experimentally in humans, we employed a murine model toexamine the hypothesis.

Characterization of the Toxicity of 7 Gy Single Fraction TBI in BALB/cMice

To model potentially lethal radiation exposure, we defined a dose ofsingle fraction TBI associated with BM aplasia, GI toxicity, and a highrate of early mortality in BALB/c mice. A single fraction of 7 Gy wasassociated with 95-100% mortality by 30 days (LD_(95/30)) in 12 week oldBALB/c (FIG. 2A). The lethality of 7 Gy exposure was reproduciblyobserved in each of the ensuing mitigation experiments: only 5/90 7 Gyirradiated mice (5.5%) survived to D30 and median survival in separateexperiments ranged from 12-15 days. Following 7 Gy TBI, small bowelepithelial apoptosis assessed by histopathology was maximal at D3, andparalleled a fall in plasma citrulline levels, which are directlyproportional to functional GI enterocyte mass (30). (FIG. 2B). Both GImucosal findings improved by D6-9. Endotoxemia was also detectable by D3and persisted until spiking higher just prior to death (FIG. 7). By D3,the BM was aplastic (FIG. 2C), with a concurrent fall of nearly 2 logsin BM mononuclear cell (MNC) content, including a decrement inhematopoietic stem (LSK, Lin⁻ Sca-1⁺ c-Kit⁺) and progenitor (LK, Lin⁻Sca-1⁻ c-Kit⁺) cells (FIG. 2D, E, F).

The degree of mucosal injury, inflammation and toxicity experiencedduring human HSCT has been related to the intensity of myeloablation(31). To ensure the model was adequately myeloablative, we compared theeffects of 7 and 6.5 Gy on hematopoiesis. Although histologic aplasiawas identical at D3 regardless of TBI dose, mice had significantlygreater BM MNC, LK and LSK 3, 10 and 15 days after 6.5 Gy than after 7Gy (FIG. 2D, E, F). No significant recovery was observed in the 7 Gycohort by D15. Subsequent mitigation experiments were performed afterexposure to 7 Gy.rBPI₂₁ and Enrofloxacin (ENR) Administration Markedly DecreaseTBI-Related Mortality

The combination (rBPI₂₁/ENR) of rBPI₂₁ and oral ENR, a fluoroquinoloneantibiotic analogous to ciprofloxacin, initiated 24 hours after anLD_(95/30)TBI dose of 7 Gy and continued through D30, produced astatistically significant improvement in D30 survival of mice (FIG. 3).In a composite analysis of 2 replicate experiments (aggregate n=50mice/arm), survival of the rBPI₂₁/ENR group exceeded that of the VEH/ENR(VEH denotes the formulation buffer for rBPI₂₁), as well as the ENR or 7Gy alone groups (<0.0001, 0.03 and <0.0001, respectively, by pair-wiseMantel-Cox log-rank). Only two deaths in 36 at-risk rBPI₂₁/ENR treatedmice occurred after two weeks whereas losses in the other groups overthis interval ranged from 38-79% of at risk animals. D30 survival after7 Gy was not improved by either rBPI₂₁ (1/30 survivors) or its VEH (1/30survivors) alone. Thus, rBPI₂₁ monotherapy was not pursued as amitigation strategy at an LD_(95/30) TBI dose.

rBPI₂₁ has a 3 hour half-life in mice when administered by IV bolus orsubcutaneous (SC) injection. As optimal continuous or q6hr IV or SCinjection regimens were not feasible, we elected to use twice daily SCadministration, initiating all treatments 24 hours after TBI andcontinuing through D30. As illustrated in FIG. 3, the control for therBPI₂₁/ENR regimen, VEH/ENR, was associated with worse 30 day survivalthan oral ENR alone (p=0.0002, by pair-wise Mantel-Cox log-rank for 2replicate experiments with combined n=50/arm), suggesting thatrepetitive handling and local skin trauma entailed in SC administrationwere associated with significant toxicity. Local skin injury was readilyobserved in irradiated mice repetitively injected with rBPI₂₁ or VEH incomparison to irradiated mice treated with ENR alone (FIG. 8).

A curtailed schedule, stopping injection after 14 days (denotedrBPI₂₁(14) and VEH(14) in FIG. 3B) was explored. Reasoning that oralantibiotic treatment could be more readily deployed in a mass-casualtysetting, the ENR schedule was not changed. rBPI₂₁ (14)/ENR provided thesame survival advantage as the longer schedule (FIG. 3B). Six irradiatedmice that had received rBPI₂₁(14)/ENR were followed beyond D30, and fiveof the six mice remained alive and healthy-appearing at D131.

rBPI₂₁/ENR Administration Mitigates Hematopoietic Toxicity after TBI

To characterize effects on hematopoiesis, we enumerated BM MNC retrievedfrom flushed BM cavities (FIG. 4). At D10, all irradiated groups,regardless of treatment, exhibited fewer BM MNC than unirradiated,age-matched controls (p<0.0001). However BM MNC content wassignificantly greater in the rBPI₂₁/ENR treated mice than in micereceiving either 7 Gy alone or with ENR or VEH/ENR (p=0.0003, 0.001 and<0.0001, respectively). This same pattern was repeated on D15 and D19,as the rBPI₂₁/ENR treated mice consistently had statisticallysignificantly greater BM MNC content than the other groups (FIG. 4).Only rBPI₂₁/ENR treatment was associated with consistently greater BMMNC content than observed after 7 Gy alone.

This rapid increase in BM MNC was reflected in the cellularity of BMassessed by histopathology on D19 (FIG. 4). rBPI₂₁/ENR treatedirradiated mice demonstrated well-recovered BM cellularity, ranging from80-90%, whereas that of the 7 Gy alone, ENR, and VEH/ENR treated micewas estimated at 20, <5, and 10-50%, respectively. Trilineagehematopoiesis was observed in all mice with sufficient cellularity, anda subset of mice, most notably those receiving rBPI₂₁/ENR, had myeloidpredominance and increased megakaryopoiesis (FIG. 9). By D30, allsurviving mice demonstrated improved cellularity, as previouslydescribed in murine TBI survivors (32). However, rBPI₂₁/ENR treated micedemonstrated more robust cellularity with significantly greater BM MNCthan ENR or VEH/ENR (FIG. 5). Recovering cellularity was also seen inthe more limited pool of 7 Gy survivors at D30.

While the number of LSK and LK remained below the age-matchedunirradiated controls at each time point, administration of rBPI₂₁/ENRwas associated with increased numbers of LSK and LK BM cells in thefirst weeks after TBI (FIG. 6). The absolute number of both LSK and LKper hind limb in rBPI₂₁/ENR mice was significantly greater than that in7 Gy alone, ENR or VEH/ENR treated mice at D10 and D19. No othertreatment was associated with a difference from the untreated irradiatedgroup. At D30, there was no difference among surviving mice betweentreatment groups or between treatment and normal controls.

BM changes correlated with changes in peripheral blood counts. Theeffects of 7 Gy on PB counts could be seen in virtually everyhematologic parameter measured (Table 1). rBPI₂₁/ENR treatment wasassociated with greater recovery of white blood cell (WBC), neutrophil,monocyte and platelet counts by D19 than was 7 Gy alone, ENR or VEH/ENRtreatment. In contrast to the other groups, median WBC, neutrophil andmonocyte levels of rBPI₂₁/ENR treated mice were in the normal range.Median hemoglobin was also greater in the rBPI₂₁/ENR treated mice,although this difference did not reach statistical significance. The WBCand neutrophils of rBPI₂₁/ENR treated mice remained significantlygreater than the ENR treated animals at D30, at which point there weretoo few 7 Gy alone or VEH/ENR mice for meaningful comparison to othergroups. Equivalent mitigation of hematopoietic toxicity was observedwith the shorter rBPI₂₁(14)/ENR schedule (FIGS. 12 and 13).

rBPI₂₁ Administration Markedly Increases Inflammatory-AssociatedChemokines

rBPI₂₁ treatment significantly increases inflammatory-associatedchemokines such as granulocyte colony stimulating factor (G-CSF) (FIG.14), murine keratinocyte chemoattractant (murine KC; human homologsinclude Gro-alpha, IL-8) (FIG. 15), and monocyte chemotactic protein-1(MCP-1, also known as Chemokine (C—C motif) ligand 2 or CCL2) (FIG. 16).BPI stimulation of G-CSF and murine KC in plasma is augmented by but notisolated to prior radiation. Without intending to bound by theory, thismay represent one mechanism by which hematopoietic effects are realized.The plasma levels achieved after irradiation and rBPI administration areconsistent with infusion of pharmacologic amounts of recombinant G-CSFIV. The data demonstrates highly significant increases (1-3 logincrements) in G-CSF in irradiated mice given SC BID rBPI alone or incombination with enrofloxacin. Therefore the elevation of GCSF is afunction of the rBPI and not dependent upon the combination of BPI withENR. A less marked but statistically significant elevation of G-CSF wasalso seen in unirradiated mice given either a single injection of rBPIor BID SC injections.

An observational cohort study in patients undergoing HSCT was conductedto identify molecular and cellular changes that might be relevant to thetoxicity of myeloablative irradiation. We observed that the neutropeniaroutinely following myeloablative treatment was associated with rapiddepletion of plasma BPI, a neutrophil-derived protein with potentendotoxin neutralizing activity, (25, 26) at a time concurrent withendotoxemia. These changes paralleled cellular (mCD14, TLR4 surfacelevels), plasma (IL-6) and physiologic (fever) alterations consistentwith increased systemic endotoxin activity (24, 25). We also observedthat lower plasma BPI concentrations at the time of HSC infusion (D0)correlated with more delayed myeloid engraftment, suggesting thatendotoxin might directly or indirectly exert some negative influence onHSC at time of infusion and for a period thereafter. The ability ofexogenous BPI supplementation to mitigate radiation toxicity in humansexposed to TBI doses that produce mucosal injury, endotoxemia andprolonged BM aplasia was then explored. Using an LD_(95/30) singlefraction myeloablative TBI model in BALB/c mice, we demonstrated that acombination of rBPI₂₁ and ENR, initiated 24 hours after radiationexposure was associated with survival of two-thirds or more of theanimals (p<0.0001). We selected rBPI₂₁ and a fluoroquinolone antibioticas an immediately actionable strategy; both agents have biologicactivity and highly favorable safety profiles in healthy and ill humans,including those with multi-organ compromise (33-43). rBPI₂₁ alone didnot improve survival, whereas ENR alone provided some survival benefit.Mitigation effects of fluoroquinolones alone have been variable,potentially related to differences including the animal model andtreatment design (15, 16). In this study, the survival benefit of ENRtreatment was significantly less than that of rBPI₂₁/ENR, despite therepetitive injury of the injection regimen. Moreover, irradiated animalstreated with VEH/ENR or ENR were characterized by delayed recovery ofevery hematopoietic parameter examined. Only rBPI₂₁/ENR was consistentlyassociated with both improved survival and more rapid and completehematopoietic recovery. These may be related findings as suggested bysurvival of 97% of rBPI₂₁/ENR treated animals after the reconstitutionof near normal BM cellularity and PB counts documented on D19.

The contributions of hematopoietic syndrome to the morbidity andmortality of ARS in humans (1, 2, 4, 44-46) underscores the relevance ofthe observed effects of rBPI₂₁/ENR on hematopoiesis. Allogeneic HSCTmitigates BM failure resulting from myeloablation, (5) but it isunlikely that resource-intensive HSCT could be implemented rapidly orsuccessfully during a mass radiation exposure (44-46). Multiple agents(47), including fluoroquinolones (16) and the TLR agonists flagellin(19, 21) and endotoxin (17), provide some radioprotection in animalmodels if administered prior to TBI. In contrast, few agents havedemonstrated efficacy when administered after radiation (i.e. radiationmitigation) and efficacy has generally been dependent uponadministration within minutes to hours after radiation exposure.Unfortunately, such rapid deployment of a mitigation strategy isunlikely, making strategies that can be delayed for 24 hours or morehighly desirable.

There is no established radiation dosimetry technology that canaccurately triage exposed individuals and determine those most likely tobenefit from mitigation treatment, nor is there a human therapeuticapplication of TBI without HSC support in which to study the efficacyand toxicity of radiation mitigation agents. These limitations highlightthe importance of selecting strategies unlikely to produce toxicity ineither minimally affected or critically-ill populations. The componentsof the strategy studied here meet this criterion. The human equivalentof ENR, a veterinary fluoroquinolone, is ciprofloxacin which was FDAapproved in 1987. Fluoroquinolones have excellent oral bioavailability,are well-tolerated and have been widely and safely used aftermyeloablative chemoradiotherapy (42, 43). rBPI₂₁ is available in asoluble form with demonstrated stability when stored at 2-8° C.facilitating stockpiling. It may be administered SC, IV and IP and inanimals has shown efficacy in an intranasal form. In addition toefficacy in animal models of pure endotoxemia and Gram-negativebacteremia, rBPI₂₁ can abrogate the signs and symptoms of endotoxemia inhumans and decrease or eliminate associated cytokine dysregulation andcoagulopathy (38, 39). No significant toxicity has been seen in PhaseI-III trials enrolling >1100 normal volunteers and critically illpatients, including infants and subjects with meningococcemia orundergoing major operative procedures (33-41). In a pilot experience(n=4), we have also administered rBPI₂₁ to patients receiving TBI aspart of myeoloablative HSCT without any attributable toxicity (FIG. 11).In aggregate, these data suggest rBPI₂₁/ENR could be safely administeredto individuals with poorly documented degrees of radiation exposure.

Increased global concerns about radiation injury consequent to naturaldisasters, nuclear conflict or terrorism or as an untoward consequenceof intentional medical exposure led us to investigate whethersupplemental BPI could be translated to an effective radiationmitigation strategy. Our data suggest that the combination of rBPI₂₁ anda fluoroquinolone antibiotic, started as late as 24 hours after apossibly lethal radiation exposure, has the potential to both improvesurvival and limit the scope and duration of requisite supportive care.Given the relatively low sensitivity of mice to endotoxin in comparisonto humans, the sub-optimal dosing, and the repetitive stress andinflammatory response of SC injection in this model, our results mayunderestimate the potential benefit of this combination. The observedefficacy of treatment initiated 24 hours post-TBI is shared by few otherapproaches and weighs heavily in its favor. The human safety record ofrBPI₂₁ and fluoroquinolones provides a platform for rapid adoption thatis particularly compelling given the obligate overtreatment resultingfrom current limitations of radiation dosimetry and affords decreasedlikelihood of unanticipated side-effects. In addition to neutralizingendotoxin, rBPI₂₁ exerts antibacterial activity that, in addition to theantibiotic activity of a fluoroquinolone, could potentially curtailfurther polypharmacy and minimize emergence of resistant species inradiated individuals with numerous reasons for infection. This reportprovides a foundation for pursuing the mechanisms by which rBPI₂₁impacts radiation toxicity. While optimization of the formulation,dosing regimen, and length of therapy for rBPI₂₁ or like agents issimilarly desirable, consideration of rBPI₂₁ approval for thisindication and subsequent stockpiling for combined mitigation therapy inthe case of radiation disaster appears warranted.

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We claim:
 1. A method for mitigating hematopoietic tissue injury in asubject resulting from exposure to chemotherapy, the method comprising:administering to a subject in need thereof bactericidal/permeabilityincreasing protein (BPI) and/or one or more of its congeners in anamount effective to mitigate hematopoietic tissue injury caused bychemotherapy exposure of the subject, wherein the subject has a decreasein bone marrow function or a decreased blood cell count as compared toexpected normal levels.
 2. The method of claim 1, wherein thehematopoietic tissue injury comprises hematopoietic toxicity.
 3. Themethod of claim 1, wherein the chemotherapy exposure results frommyeloablative conditioning for hematopoietic stem cell transplant in thesubject.
 4. The method of claim 1, wherein one or more of the BPIcongeners is selected from the group consisting of rBPI₂₁, rBPI₂₃,rBPI₅₀, rBPI(10-193)ala¹³² and a N-terminal fragment of BPI having anapproximate molecular weight of from about 20 kD to about 25 kD.
 5. Themethod of claim 1, further comprising administering an antibiotic. 6.The method of claim 5, wherein the antibiotic is a quinolone antibiotic.7. The method of claim 6, wherein the quinolone antibiotic is selectedfrom the group consisting of moxifloxacin, ciprofloxacin, levofloxacin,garenoxacin, and delafloxacin.
 8. The method of claim 1, wherein BPIand/or its congeners is administered between 1 day before and 2 daysafter exposure of the subject to the chemotherapy exposure.
 9. Themethod of claim 8, wherein BPI and/or its congeners is administeredwithin 48 hours after the chemotherapy exposure.
 10. The method of claim1, wherein BPI and/or its congeners is administered orally,intravenously, subcutaneously, or pulmonarily.
 11. The method of claim1, wherein BPI and/or its congeners improves the likelihood of survivalof the subject.
 12. The method of claim 1, wherein BPI and/or itscongeners restores blood cell counts to normal levels.
 13. The method ofclaim 1, wherein the hematopoietic tissue is bone marrow.
 14. The methodof claim 13, wherein the subject has a hematopoietic deficiency in oneor more hematopoietic cell types or lineages.
 15. The method of claim14, wherein the hematopoietic deficiency is one or more of: lymphopenia,myelopenia, leukopenia, neutropenia, erythropenia, megakaryopenia, adeficiency in platelets, a deficiency in monocytes, a deficiency inlymphocyctes, a deficiency in erythrocytes, deficiency in neutrophils, adeficiency in T cells, a deficiency in granulocytes, and a deficiency indendritic cells.
 16. The method of claim 1, wherein the subject hascancer and the BPI and/or its congeners is administered at least 1 hourfollowing chemotherapy, but not more than 72 hours followingchemotherapy.
 17. A method for mitigating deficiency in platelets in asubject resulting from exposure to chemotherapy, the method comprising:administering to a subject in need thereof bactericidal/permeabilityincreasing protein (BPI) and/or its congeners in an amount effective tomitigate deficiency in platelets caused by the chemotherapy exposure tothe subject, wherein the BPI or its congeners is administered between 1day before and 2 days after exposure of the subject to the chemotherapy.18. The method of claim 17, wherein one or more of the BPI congeners isselected from the group consisting of rBPI₂₁, rBPI₂₃, rBPI₅₀,rBPI(10-193)ala¹³² and a N-terminal fragment of BPI having anapproximate molecular weight of from about 20 kD to about 25 kD.
 19. Themethod of claim 17, further comprising administering an antibiotic. 20.The method of claim 19, wherein the antibiotic is a quinoloneantibiotic.
 21. The method of claim 20, wherein the quinolone antibioticis selected from the group consisting of moxifloxacin, ciprofloxacin,levofloxacin, garenoxacin, and delafloxacin.